The control of transcription depends on DNA-bound regulatory proteins and their interactions. The proposed research is aimed at examining two important classes of protein-protein interactions, dimerization and cooperativity, using several prokaryotic model systems. Genetic methods will be employed to identify the molecular determinants that govern the specificity of these interactions. Many transcriptional regulators in both prokaryotes and eukaryotes function in dimeric form, either as homodimers or heterodimers, and in some cases the same protein can associate with alternative partners, carrying out distinct regulatory functions depending on its partner. Thus, the proper biological activity depends on the abilities of potential partners to discriminate amongst one another so that only the correct dimeric species are formed. The proposed research will address the problem of dimerization specificity with an analysis of the dimerization specified of the E. coli cAMP receptor protein (CRP) and its homologue FNR. Many transcriptional regulators also bind cooperatively to the DNA in either homotypic or heterotypic combinations. The assembly of eukaryotic regulatory complexes, in particular, typically involves multiple DNA- bound proteins that participate in a variety of weak protein-protein interactions. Cooperativity is both a mechanism for generating sensitive biological switches by providing a steep dose-response cure, as well as a means by which multiple regulatory inputs can be integrated. The proposed research will probe the nature of the underlying protein- protein interactions with a detailed analysis of model cooperativity interface, that of the bacteriophage lambda repressor and its homologues. A general objective of the proposed research is the development of new genetic methods that can be widely applied to the study of biologically important protein-protein interactions. A recently developed E. coli- based two-hybrid assay will be tested for its efficacy in detecting interacting proteins encoded in complex libraries. This assay will also be applied to the study and manipulation of a phosphorylation-dependent protein-protein interaction involving the eukaryotic transcription factor CREB and its CREB and its co-activator CBP.